Klaus Koren

Precipitation as a simple and versatile method for preparation of optical nanochemosensors.

Optical nanosensors for such important analytes as oxygen, pH, temperature, etc. are manufactured in a simple way via precipitation. Lipophilic indicators are entrapped into nanobeads based on poly(methyl methacrylate), polystyrene, polyurethanes, ethylcellulose, and other polymers. Charged groups greatly facilitate formation of the small beads and increase their stability. Sensing properties of the beads can be tuned by choosing the appropriate indicator. Nanosensors for carbon dioxide and ammonia are found to be cross-sensitive to pH if dispersed in aqueous media. These nanobeads are successfully employed to design bulk optodes. Nanochemosensors with enhanced brightness via light-harvesting and multi-functional magnetic nanosensors also are prepared.

Small molecule phosphorescent probes for O2 imaging in 3D tissue models

Monitoring of oxygenation is important for physiological experiments investigating the growth, differentiation and function of individual cells in 3D tissue models. Phosphorescence based O2 sensing and imaging potentially allow this task; however, current probes do not provide the desired bio-distribution and analytical performance. We present several new cell-penetrating phosphorescent conjugates of a Pt(ii)-tetrakis(pentafluorophenyl)porphine (PtPFPP) dye produced by click-modification with thiols, and perform their evaluation as O2 imaging probes for 3D tissue models. The hydrophilic glucose (Pt-Glc) and galactose (Pt-Gal) conjugates demonstrated minimal aggregation and self-quenching in aqueous media, and efficient in-depth staining of different cell types and multi-cellular aggregates at working concentrations ≤10 μM. The Pt-Glc probe was applied in high-resolution phosphorescence lifetime based O2 imaging (PLIM) in multi-cellular spheroids of cancer cells (PC12), primary neural cells (neurospheres) and slices of brain tissue, where it showed good analytical performance, minimal effects on cell viability and appropriate responses to O2 with phosphorescence lifetimes changing from 20 μs in air-saturated to 57 μs under deoxygenated conditions. In contrast, mono- and tetra-substituted oligoarginine conjugates of PtPFPP showed marked aggregation and unstable photophysical properties precluding their use as O2 sensing probes.

Strongly Phosphorescent Iridium(III)-Porphyrins - New Oxygen Indicators with Tuneable Photophysical Properties and Functionalities.

Synthesis and characterization of four iridium(III)–octaethylporphyrins and a π-extended iridium(III)–benzoporphyrin are presented. Strong room-temperature phosphorescence was observed for all of the complexes with quantum yields of up to 30 %. Axial ligands were introduced to tune the photophysical properties and the solubility. Complexes bearing lipophilic ligands such as pyridine or N-(n-butyl)imidazole were incorporated into polystyrene to obtain optical oxygen sensors. Covalent coupling of the dye is possible by introduction of ligands with binding domains (1-imidazoleacetic acid). This enabled preparation of a water-soluble oxygen probe (by staining bovine serum albumin) and a trace oxygen sensor (by coupling to amino-modified silica gel).

Complexes of IrIII-Octaethylporphyrin with Peptides as Probes for Sensing Cellular O2

IrIII–porphyrins are a relatively new group of phosphorescent dyes that have potential for oxygen sensing and labeling of biomolecules. The requirement of two axial ligands for the IrIII ion permits simple linkage of biomolecules by a one‐step ligand‐exchange reaction, for example, using precursor carbonyl chloride complexes and peptides containing histidine residue(s). Using this approach, we produced three complexes of IrIII–octaethylporphyrin with cell‐penetrating (Ir1 and Ir2) and tumor‐targeting (Ir3) peptides and studied their photophysical properties. All of the complexes were stable and possessed bright, long‐decay (unquenched lifetimes exceeding 45 μs) phosphorescence at around 650 nm, with moderate sensitivity to oxygen. The Ir1 and Ir2 complexes showed positive staining of a number of mammalian cell types, thus demonstrating localization similar to endoplasmic reticulum and ATP‐ and temperature‐independent intracellular accumulation (direct translocation mechanism). Their low photo‐ and cytotoxicity allows intracellular oxygen to be probed.

Optical sensor nanoparticles in artificial sediments--a new tool to visualize O2 dynamics around the rhizome and roots of seagrasses.

Seagrass communities provide important ecosystems services in coastal environments but are threatened by anthropogenic impacts. Especially the ability of seagrasses to aerate their below-ground tissue and immediate rhizosphere to prevent sulfide intrusion from the surrounding sediment is critical for their resilience to environmental disturbance. There is a need for chemical techniques that can map the O2 distribution and dynamics in the seagrass rhizosphere upon environmental changes and thereby identify critical stress thresholds of e.g. water flow, turbidity, and O2 conditions in the water phase. In a novel experimental approach, we incorporated optical O2 sensor nanoparticles into a transparent artificial sediment matrix consisting of pH-buffered deoxygenated sulfidic agar. Seagrass growth and photosynthesis was not inhibited in the experimental setup when the below-ground biomass was immobilized in the artificial sulfidic sediment with nanoparticles and showed root growth rates (∼ 5 mm day(-1)) and photosynthetic quantum yields (∼ 0.7) comparable to healthy seagrasses in their natural habitat. We mapped the real-time below ground O2 distribution and dynamics in the whole seagrass rhizosphere during experimental manipulation of light exposure and O2 content in the overlaying water. Those manipulations showed that oxygen release from the belowground tissue is much higher in light as compared to darkness and that water column hypoxia leads to diminished oxygen levels around the rhizome/roots. Oxygen release was visualized and analyzed on a whole rhizosphere level, which is a substantial improvement to existing methods relying on point measurements with O2 microsensors or partial mapping of the rhizosphere in close contact with a planar O2 optode. The combined use of optical nanoparticle-based sensors with artificial sediments enables imaging of chemical microenvironments in the rhizosphere of aquatic plants at high spatiotemporal resolution with a relatively simple experimental setup and thus represents a significant methodological advancement for studies of environmental impacts on aquatic plant ecophysiology.

Magnetically remote-controlled optical sensor spheres for monitoring oxygen or pH.

Magnetic sensor macrospheres (MagSeMacs), i.e., stainless steel spheres coated with optical chemical sensors, are presented as an alternative to existing optical sensor patches and fiber-optical dip-probes. Such spheres can either be reversibly attached to the tip of an optical fiber (dip-probe) or trapped inside a vessel for read-out through the side wall. Moving the magnetic separator at the exterior enables measurements at varying positions with a single sensor. Moreover, the sensor’s replacement is rapid and contactless. We measured dissolved oxygen or pH in stirred liquids, rotating flasks, and 24-well plates with a SensorDish-reader device for parallel cell culture monitoring. In these applications, MagSeMacs proved to be advantageous over conventional sensor patches and magnetic optical sensor particles because of their magnetism, spherical shape, reflectance, and size. These properties resulted in strong but reversible fixation, magnetic remote-controllability, short response times, high signal intensities, and simplified handling.

Nanoparticle‐based measurements of pH and O2 dynamics in the rhizosphere of Zostera marina L.: effects of temperature elevation and light‐dark transitions

Seagrasses can modulate the geochemical conditions in their immediate rhizosphere through the release of chemical compounds from their below-ground tissue. This is a vital chemical defence mechanism, whereby the plants detoxify the surrounding sediment. Using novel nanoparticle-based optical O2 and pH sensors incorporated in reduced and transparent artificial sediment, we investigated the spatio-temporal dynamics of pH and O2 within the entire rhizosphere of Zostera marina L. during experimental manipulations of light and temperature. We combined such measurements with O2 microsensor measurements of the photosynthetic productivity and respiration of seagrass leaves. We found pronounced pH and O2 microheterogeneity within the immediate rhizosphere of Z. marina, with higher below-ground tissue oxidation capability and rhizoplane pH levels during both light exposure of the leaf canopy and elevated temperature, where the temperature-mediated stimuli of biogeochemical processes seemed to predominate. Low rhizosphere pH microenvironments appeared to correlate with plant-derived oxic microzones stimulating local sulphide oxidation and thus driving local proton generation, although the rhizoplane pH levels generally where much higher than the bulk sediment pH. Our data show that Z. marina can actively alter its rhizosphere pH microenvironment alleviating the local H2 S toxicity and enhancing nutrient availability in the adjacent sediment via geochemical speciation shift.

Banning toxic heavy-metal catalysts from paints: enzymatic cross-linking of alkyd resins

Alkyd resins are polyesters containing unsaturated fatty acids that are used as binding agents in paints and coatings. Chemical drying of these polyesters is based on heavy metal catalyzed cross-linking of the unsaturated fatty acid moieties. Among the heavy-metal catalysts, cobalt complexes are the most effective, yet they have been proven to be carcinogenic. Therefore, strategies to replace the cobalt-based catalyst by environmentally friendlier and less toxic alternatives are under development. Here, we demonstrate for the first time that a laccase–mediator system can effectively replace the heavy-metal catalyst and cross-link alkyd resins. Interestingly, the biocatalytic reaction does not only work in aqueous media, but also in a solid film, where enzyme diffusion is limited. Within the catalytic cycle, the mediator oxidizes the alkyd resin and is regenerated by the laccase, which is uniformly distributed within the drying film as evidenced by confocal laser scanning microscopy. During gradual build-up of molecular weight, there is a concomitant decrease of the oxygen content in the film. A new optical sensor to follow oxygen consumption during the cross-linking reaction was developed and validated with state of the art techniques. A remarkable feature is the low sample amount required, which allows faster screening of new catalysts.

Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo-Like Characteristics

ABSTRACT Alginate beads represent a simple and highly reproducible in vitro model system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h. Confocal microscopy revealed that P. aeruginosa formed dense clusters similar in size to in vivo aggregates observed ex vivo in cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2 concentration decreased rapidly and reached anoxia ∼100 μm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3− as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulated P. aeruginosa confirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulated P. aeruginosa exhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth of P. aeruginosa in alginate beads represent a simple and flexible in vivo-like biofilm model system, wherein bacterial growth exhibits central features of in vivo biofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3− as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment. IMPORTANCE Pseudomonas aeruginosa has been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation.

Seagrass-Mediated Phosphorus and Iron Solubilization in Tropical Sediments

Tropical seagrasses are nutrient-limited owing to the strong phosphorus fixation capacity of carbonate-rich sediments, yet they form densely vegetated, multispecies meadows in oligotrophic tropical waters. Using a novel combination of high-resolution, two-dimensional chemical imaging of O2, pH, iron, sulfide, calcium, and phosphorus, we found that tropical seagrasses are able to mobilize the essential nutrients iron and phosphorus in their rhizosphere via multiple biogeochemical pathways. We show that tropical seagrasses mobilize phosphorus and iron within their rhizosphere via plant-induced local acidification, leading to dissolution of carbonates and release of phosphate, and via local stimulation of microbial sulfide production, causing reduction of insoluble Fe(III) oxyhydroxides to dissolved Fe(II) with concomitant phosphate release into the rhizosphere porewater. These nutrient mobilization mechanisms have a direct link to seagrass-derived radial O2 loss and secretion of dissolved organic carbon from the below-ground tissue into the rhizosphere. Our demonstration of seagrass-derived rhizospheric phosphorus and iron mobilization explains why seagrasses are widely distributed in oligotrophic tropical waters.